Method for simultaneously improving protein crystallization success rate and crystal quality

A protein crystallization and crystal quality technology, which is applied in the field of improving the success rate of protein crystallization, can solve the problems that it is difficult to improve the crystallization success rate and crystal quality at the same time, achieve the effects of smooth changes in solution concentration, increase the success rate of crystallization, and improve the quality of crystals

Inactive Publication Date: 2014-04-02
NORTHWESTERN POLYTECHNICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] In order to solve the technical problem that the existing protein crystallization method is difficult to simultaneously improve the crystallization success rate and crystal quality, the present invention provides a method for simultaneously improving the protein crystallization success rate and crystal quality

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Example 1: Screening of lysozyme crystallization conditions.

[0023] 1. Preparation of crystallization orifice plates for adsorption-desorption materials.

[0024] The α-methyl polystyrene microspheres with a pore size of 20-50 nm are ultrasonically dispersed with alcohol to prepare a suspension with a concentration of 10 mg / mL. Add 4 μL of α-methylpolysaccharide at a concentration of 40 mg / mL to each crystallization hole of a 96-well sitting drop plate (Hampton, USA, product number: HR3-143, 96-wellsitting-drop Intelli-Plates). Styrene microspheres.

[0025] 2. Prepare lysozyme solution.

[0026] The lysozyme (Japanese Seikagaku company, product number is 837059) that is recrystallized six times is dissolved in pH=4.60, in 0.25M Tris-HCL buffer solution, then filters with 0.22 μm filter membrane, filters out impurity, makes initial concentration 40mg / mL lysozyme solution.

[0027] 3. Prepare protein crystallization solution.

[0028] Use the automatic pipetting s...

Embodiment 2

[0033] Example 2: Screening of catalase crystallization conditions.

[0034] 1. Prepare the crystallization orifice plate of the adsorption material.

[0035] Apply a small amount of vacuum ester to the intended crystallization position of each crystal hole wall of a 96-well sitting drop plate (Hampton, USA, article number HR3-143, 96-well sitting-drop Intelli-Plates), and use tweezers to Acrylonitrile carbon fiber powder adheres to the vacuum ester-coated vessel wall.

[0036] 2. Prepare catalase solution.

[0037] Dissolve catalase (US Sigma Company, product number C40) in pH 7.00, 0.025M HEPES-Na buffer solution, and then filter out impurities with a 0.22 μm filter membrane to prepare a trypsin solution with an initial concentration of 8 mg / mL .

[0038] 3. Prepare protein crystallization solution.

[0039] Use the automatic pipetting system to add the protein crystallization reagent in the Crystal Screen crystallization kit of Hampton Company to each deep crystallization...

Embodiment 3

[0044] Example 3: Screening of trypsin crystallization conditions.

[0045] 1. Prepare the crystallization orifice plate of the adsorption material.

[0046] The porous polystyrene / acrylonitrile copolymer microspheres were ultrasonically dispersed with alcohol, prepared into a suspension with a concentration of 40 mg / mL, and poured into 96-well sitting drop plates (Hampton, USA, article number HR3-143, 96-well sitting -drop Intelli-Plates), add 2 μL of polystyrene / acrylonitrile copolymer microspheres at a concentration of 60 mg / mL to the quasi-crystallization position of each crystallization hole.

[0047] 2. Prepare trypsin solution.

[0048] Trypsin (Sigma, USA, product number T8003) was dissolved in pH 7.00, 0.025M HEPES-Na buffer, and then filtered out impurities with a 0.22 μm filter membrane to prepare a trypsin solution with an initial concentration of 10 mg / mL.

[0049] 3. Prepare protein crystallization solution.

[0050] Use an automatic pipetting system to add th...

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Abstract

The invention relates to a method for simultaneously improving protein crystallization success rate and crystal quality, and is used for solving the technical problems that the traditional protein crystallization method has difficulties in simultaneously improving the crystallization success rate and the crystal quality. The technical scheme is as follows: an adsorption material with a adsorption-desorption function to protein is utilized to simultaneously form two concentration regions in a crystallization solution, and the two concentration regions comprise a high-concentration region near the adsorption material and a low-concentration region away from the adsorption region; thus the purposes that a high-concentration condition promotes protein nucleation and a low-concentration condition promotes protein crystal growth are simultaneously achieved; the adsorption material is placed on the upper part in the solution and crystal nucleates at the upper part in the solution, thereby a role of improving the crystallization success rate is played; and when the crystal grows to a size of several microns, the crystal settles to the lower part in the solution because of the gravity action, and the concentration of the solution at the lower part is low, thereby the protein crystallization success rate is increased to 40-80% from 30-67% in the prior art while the protein crystallization quality is improved.

Description

technical field [0001] The invention relates to a method for improving the success rate of protein crystallization, in particular to a method for simultaneously improving the success rate of protein crystallization and crystal quality. Background technique [0002] To carry out protein structure and function research, we must first obtain the structural information of protein molecules. Among the protein structure analysis methods, X-ray single crystal diffraction technology, as the most mature and highest resolution analysis method, undertakes more than 87% of protein structure analysis tasks. To implement this technique, high-quality protein crystals are required as samples for diffraction. However, due to the general difficulty in obtaining high-quality protein crystals, protein crystallization is one of the bottlenecks in the analysis of protein structures by X-ray single crystal diffraction technology. [0003] Literature: Thakur et al added 9 specific powders (fumed ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K1/14
Inventor 尹大川郭云珠曹慧玲张辰艳马晓亮崔超
Owner NORTHWESTERN POLYTECHNICAL UNIV
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